1. Field of the Invention
The invention relates to polypeptide antibiotics and to the identification of genetic loci associated with expression of the antibiotics. The invention particularly describes a purified lanthionine-containing antimicrobial agent, DNA encoding the protein, and methods and compositions for treatments employing the antibiotic.
2. Description of Related Art
The phenotypic ally similar group of bacteria collectively known as the mutans streptococci are considered the major etiologic agents responsible for dental caries and have been implicated as major causative agents in other infectious and transmissible diseases, such as endocarditis. The species most commonly associated with dental caries is Streptococcus mutans. Attempts to better understand the genetic determinants that contribute to the cariogenic potential of this organism, as well as its natural history as an agent of an infectious disease, have only recently been explored. For example, a molecular approach has been employed to delineate the structure, function, and regulation of a number of different enzymes including the glucosyltransferases that are involved with sugar metabolism, an important virulence factor responsible for the metabolic conversion of sucrose to extracellular polymers to form critical mass and provide for fermentable substrates (Hamada, et al., 1986).
The other two major factors thought to be involved with the pathogenicity of S. mutans are its acidogenic/aciduric properties (Caufield, et al., 1990A) and its ability to elaborate poorly characterized bacteriocin-like substances, generally known as mutacins, which may provide a selective force necessary for sustained colonization in a milieu of densely packed competing organisms found in plaque (Buchman, et al., 1988). Collectively called xe2x80x9cmutacinsxe2x80x9d these agents kill other bacteria of the same or closely related species. The mutacins are only similar in name and host producer, as their properties differ widely. Mutacins associated with plasmid-containing strains of S. mutans have been designated as either Group I or II (mutacin I and mutacin II. Therefore, the production of mutacins is one characteristic of S. mutans that appears to contribute to its ability to colonize and be sustained, particularly in the oral cavity. In this regard, mutacins may be considered virulence factors.
The mutans streptococci produce several different bacteriocin-like inhibitory substances, collectively called mutacins. To date, most remain only partially characterized (Hamada, et al., 1986; Loyola, et al., 1992). Reasons for limited success in characterization of these substances are likely to be that: 1) they are made in small quantities; 2) production occurs only under special cultivation conditions; 3) a lack of production in liquid media; and 4) difficulty in isolating these xe2x80x98mutacinsxe2x80x99 from the media.
Bacteriocins are traditionally defined as proteinaceous substances capable of exerting lethal or bactericidal effects on other bacteria within the same species or against closely related species (Tagg, et al., 1976). Among the gram-positive bacteria, however, this definition becomes somewhat less precise because inhibitory substances exhibit a wider spectrum of activity. Moreover, bacteriocins are not always proteins and do not fit the traditional definition originally intended for the better studied gram-negative bacteriocins, e.g., colicin. Recent studies involving the molecular characterization and sequencing of several gram-positive bacterial inhibitory polypeptides, including nisin, epidermin, pep5 and subtilin, among others, reveal ribosomally translated, but post-translationally processed peptides having modified amino acids and thioether linkages. Collectively, these polypeptide antibiotics are termed lantibiotics or lanthionine-containing antibiotics (Allgaier, et al., 1986; Buchman, et al., 1988). It is now clear that mutacin II group belongs to the lanthionine-containing family of antibiotics.
A few characteristics of certain mutacins have been determined. xe2x80x9cMutacinxe2x80x9d activity appears to be associated with the presence of a 5.6 kb plasmid in S. mutans; however mutacin production in plasmid-containing strains of mutans streptococci is not plasmid-encoded (Caufield, et al., 1990A). Among strains of S. mutans harboring a 5.6 kb plasmid, at least two distinct, but closely related mutacin producing/immunity groups exist. Efforts have consequently focused on locating and characterizing the chromosomal locus responsible for mutacin expression. Initial attempts to identify the gene(s) responsible for mutacin expression employed transpositional mutagenesis using transposon Tn916 as the mutagen (Caufield, et al., 1990B). Five different chromosomal loci associated with mutacin expression have been identified.
Unfortunately, very little is known about the many described extracts with xe2x80x9cmutacinxe2x80x9d activity, such as their composition and under what conditions they are made. Because the compositions investigated were made in small quantities and only under certain poorly defined conditions, attempts to partially isolate and biochemically characterize xe2x80x9cmutacinsxe2x80x9d have yielded various results. Past studies, primarily using crude cell-free extracts, led to the description of mutacins with different molecular weights and differential sensitivity to pH and digestive enzymes (Caufield, et al. (1985, 1990A, 1990B), Loyola-Rodriguez, et al. (1992), Delisle (1986), Pinto Alves, et al. (1992)). The relevance and usefulness of these results are particularly confounded by the lack of purity of the compositions.
A recognized need is the development of antimicrobials effective against both a broad and a defined, but limited range of microorganisms. The present state of knowledge concerning mutacin-type antibiotics suggests that these compounds have potential as a class of antibacterials. However, owing to the lack of purity of the different mutacins and the inability to obtain such compounds in sufficient quantities, specific mutacins have yet to be clearly identified or developed for further use.
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing purified mutacin compositions and methods for the treatment of microbial infections in animals, including humans. The mutacin antimicrobials are isolated from Streptococcus mutans and show activity against several species of gram-positive bacteria. The invention also provides for the molecular cloning of the mutacin gene.
As used herein, xe2x80x9cmutacinxe2x80x9d is used to designate an antimicrobial agent isolatable from S. mutans and characterized by the properties disclosed herein. These properties include an apparent molecular weight of about 2,500 Da as determined by HPLC-size exclusion chromatography, thermostability up to 100xc2x0 C., resistance to a pH range of about 4-10, an isoelectric point of  greater than 8.4 and insolubility in water-immiscible organic solvents such as CHCl3. The term xe2x80x9cisolatable fromxe2x80x9d as used herein, is intended to describe the various sources from which a mutacin in accordance with the present invention may be obtained. Appropriate. sources are considered to be virtually all S. mutans species, with T8 and UA96 being particularly preferred, and also recombinant host cells engineered to express mutacin.
It is recognized that the literature describes impure compositions containing several mutacins elaborated by S. mutans and related species. However these xe2x80x9cmutacinxe2x80x9d containing compositions are different from the mutacin described herein in several important respects, including physical and biological properties, in addition to the significant factor of their purification.
The first area of disparity is in the predicted molecular weight characteristics of xe2x80x9cmutacinsxe2x80x9d. Parrot et al. (1990) describe four xe2x80x9cmutacin extractsxe2x80x9d with Mr""s ranging in the about 3500 Da to  less than 12,000 Da as measured by dialysis, while Delisle (1986) describes a mutacin with an Mr of between 3500 and 6000 Da also by dialysis, and Loyola-Rodriguez (1992) demonstrates a molecular weight of 6500 Da by SDS-PAGE electrophoresis.
Another area of significant disparity in the mutacin field relates to the relative purity of the samples tested. Crude bacterial extracts or supernatants have been described by Pinto-Alves (1992), Parrot (1990), and Delisle (1986) to contain xe2x80x9cmutacinxe2x80x9d activity. These crude preparations differentially inhibited the growth of gram-positive bacteria, however, the exact component(s) responsible for this activity are unknown. Loyola-Rodriguez, et al. have shown a somewhat purified xe2x80x9cmutacinxe2x80x9d from S. sobrinus isolated through a series of chromatographic steps and having an Mr of 6500 Da as measured by SDS-PAGE. However, this relatively purified mutacin did not exhibit bacteriocidal activity against Actinomyces sp., Staphylococcus sp., Lactobaccilus sp., or Escherichia coli. 
Since the original description of a xe2x80x9cmutacinxe2x80x9d over 10 years ago, a purified mutacin from S. mutans has eluded production, purification, and sequencing. Although some preliminary characterization of mutacins has been reported, the present invention describes for the first time the ability to produce a Streptococcus mutans mutacin in liquid culture. Also disclosed are advantageous methods for purifying mutacin in sufficient quantities to enable characterization of its biochemical properties and, indeed, in enough purity for protein sequencing.
The term xe2x80x9cpurified mutacinxe2x80x9d as used herein, is intended to refer to a protein composition, isolatable from S. mutans, such as T8 and UA96, wherein the mutacin is purified to a substantial degree relative to its naturally-obtainable state, i.e., in this case, relative to its purity within a bacterial extract or growth supernatant. A purified mutacin therefore also refers to a mutacin polypeptide, significantly free from the environment in which it naturally occurs.
Generally, xe2x80x9cpurifiedxe2x80x9d will refer to a composition which has been subjected to fractionation to remove several non-antimicrobial components, and which composition substantially retains its bactericidal or bacteriostatic activity. Purified, in this sense, usually refers to a composition in which the mutacin forms the major component of the composition, such as constituting about 90% of the proteins in the composition or more. The purified mutacin will typically be purified about 60- or 84-fold, i.e., to such a degree so that it has a specific activity of about 110,000 bacteriocidal units per milligram of total protein (BU/mg), or preferably, between about 110,000 and 150,000 BU/mg, and most preferably it will have a specific activity of about 150,000 BU/mg. Higher purity can be obtained by methods such as HPLC, in addition to the usual purification steps.
The isolated antimicrobial mutacin polypeptides of the invention are generally characterized as having substantial bacteriocidal activity. As used herein the term xe2x80x9csubstantial bacteriocidal activityxe2x80x9d describes significant mutacin-directed bacterial killing, as measured by any one of a variety of assays. Bacteriocidal activity may be advantageously examined and quantified by the deferred antagonism technique on trypticase soy-yeast agar (TSAY) using S. sobrinus OMZ176 or S. sanguis Ny101 as sensitive indicators (Parrot, et al., 1990). A unit of bacteriocidal activity (BU), present in liquid culture, is described as the lowest titer yielding a clear lytic-like zone of inhibition having clear edges and greater than 10 mm in diameter. Chikindas et al. describes arbitrary units (AU) of activity, which are essentially identical to bacteriocidal activity units.
In certain embodiments, purified mutacin may be characterized a s being the predominant band on an SDS-PAGE gel as detected, e.g., Coomassie Blue or silver staining. Most preferably, the purified mutacin is the only band detected on an SDS-PAGE gel stained with either Coomassie blue or silver stain, according to the amount to be detected.
The mutacins of the present invention have an apparent Mr of about 2,500 Da as determined by high performance liquid chromatography using a size-exclusion column. This is a useful defining parameter which may be determined using the routinely employed technique of column chromatography. However, the highly purified mutacins may be found to have a slightly different exact molecular weights, depending on the technique used, such as the molecular weight of 3,244.64xc2x11.15 Da determined by ion spray mass spectroscopy for the T8-derived mutacin as determined from the m/z values, wherein z is equal to 2, 3, or 4 for m / z values 1623, 1083 and 812, respectively (see FIG. 4). Furthermore, the mutacin is thermostable, as measured by, e.g., incubating an agar plug at 100xc2x0 C. for 30 minutes and quantitating bacteriocidal activity. The mutacin is stable over a pH range of 4-10, as measured by incubating the purified mutacin for 24 hours in appropriate 0.1 M buffers, e.g., such as acetate, phosphate or glycine buffers, at room temperature, and tested for bacteriocidal activity. The mutacin has an isoelectric focusing point of xe2x89xa78.4 and has limited solubility in aqueous buffers but is solubilized better in 70% ethanol, 8M urea (such as 70% ethanol or 8M urea), and guanidine-water solutions.
The mutacin has the following composition of amino acids per molecule: Arg2, Asn2, Glu1, Gln2, Gly, His, Ile, Met, Pro, Trp3, Phe, Tyr, Val3, Xaa2, Yaa, Zaa, wherein Xaa represents lanthionine, Yaa represents xcex2-methyllanthionine and Zaa represents a dihydro amino acid. The mutacins of the invention may also be characterized by the presence of certain sequences. For example, by including the N-terminal sequence Asn Arg Trp Trp Gln Gly Val Val (SEQ ID NO:1), or by including an internal sequence Met Asn Xaa Trp Gln His (SEQ ID NO:2).
The development of a growth medium which permits the purification of mutacin from liquid culture is an important factor to the invention. Chemically defined media (van der Rijn et al. (1980)) supplemented with yeast extract and soy bean trypticase are the preferred media for use in connection with mutacin purification. Accordingly where the term, growing a culture of a bacteria that produces the polypeptide, is used, this preferably means growing or culturing the bacteria in media comprising a chemically defined media supplemented with yeast extract and soy bean trypticase.
The mutacin polypeptides of the present invention may also be characterized as being isolatable by a method employing ultrafiltration and selective precipitation. More specifically, mutacin polypeptides may be isolated by a process including the steps of: growing a culture of a bacteria that produces the polypeptide; filtering the culture through a porous membrane filter and collecting the flowthrough; sequentially passing the flowthrough through ultrafiltration membranes; collecting and retaining the filtrate of the final ultrafiltration step; mixing the retained filtrate with a water-immiscible organic solvent, such as acetone, benzene, ether, chloroform, ethyl acetate, methylene chloride, carbon tetrachloride, or chloroform, thereby forming a precipitate; washing the precipitate thus formed and solubilizing the precipitate using a chaotropic agent, such as guanidine, guanidinium or urea.
To prepare purified mutacin in accordance with the most preferred method of the present invention, one would first grow S. mutans T8 in supplemented growth media and then separate insoluble bacterial debris through a porous membrane, preferably a 0.45 xcexcm diameter porous membrane. The collected flowthrough would be further subjected to sequential ultrafiltration through membranes with molecular weight cut-offs of, e.g., 100-, 10-, and 1-kDa. The retained filtrate of the final ultrafiltration step is collected and a precipitate is formed by mixing this final filtrate with a water immiscible organic solvent, and the solvent evaporated. The water immiscible organic solvent preferably is chloroform. The precipitate is then washed with water or an aqueous buffer such as phosphate buffered saline, the like. The precipitate is solubilized with a chaotropic agent, such as guanidine, guanidinium, or urea, with 8M urea being most preferred. The dissolved mutacin-chaotrope mixture is finally precipitated with 20 volumes of water. The substantially purified mutacin polypeptides has bacteriocidal activity against gram-positive bacteria, as exemplified by exhibiting bacteriocidal activity against members of the Actinomyces, Bacillus, Clostridium, Mycobacterium, Staphylococcus or Streptococcus species.
The polypeptide may also be purified by solvent-based thin-layer chromatography on TLC-coated Silicagel developed with a solvent comprised of methanol:water (7:3). The polypeptide containing portion may be detected by UV and the presence of mutacin antimicrobial activity confirmed using a biological sensitivity assay.
In a preferred embodiment the mutacin is purified from the supernatant or bacterial extract of Streptococcus mutans bacteria. In a more preferred embodiment the Streptococcus mutans bacteria is of the UA96 or T8 strain. In an alternative embodiment the polypeptide may be purified from a recombinant host cell which incorporates DNA, such as an expression vector, which encodes the mutacin. A preferred host cell for this use is E. coli, with an E. coli strain that is recA being more preferred.
Various methods for quantifying the degree of purification of the mutacin will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the relative purity of the polypeptide within a fraction by SDS/PAGE analysis. One preferred method for assessing the purity of a mutacin fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial bacterial extract, and to thus calculate the degree of purity, herein assessed by a xe2x80x9c-fold purification numberxe2x80x9d.
The actual units used to represent the amount inhibitory activity is, of course, dependent upon the particular assay technique chosen to follow the purification. Preferred is an assay based upon the deferred antagonism technique. One unit of activity in the assay used herein is defined as the lowest titer of activity that yields a lytic-like zone of inhibition having clear edges and being greater than 10 mm in diameter. However, using other assays, the definition of a unit of activity would naturally vary.
As is generally known in the art, to determine the specific activity, one would calculate the number of units of activity per milligram of total protein. In the purification procedure, the specific activity of the starting material, i.e., of the bacterial extract, would represent the specific activity of the mutacin in its natural state. At each step, one would generally expect the specific activity of the mutacin to increase above this value, as it is purified relative to its natural state. The use of xe2x80x9cfold purificationxe2x80x9d is advantageous as the purity of an antimicrobial fraction can thus be compared to another despite any differences which may exist in the actual units of activity or specific activity.
It is contemplated that the antimicrobial mutacin of the present invention will preferably exhibit a specific activity of about 100,000 or 110,000 BU/mg, and may be purified up to a level of about 150,187 BU/mg of total protein. More preferably, the mutacins are particularly characterized as those being between about 60-fold purified and about 84-fold purified, with respect to the starting composition.
The most preferred method for purifying mutacin is that of filtration and organic solvent precipitation, however, various techniques suitable for use in protein purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
The preferred purification method disclosed herein above contains several steps and represents the best mode presently known by the inventors to prepare a substantially purified mutacin. This method i s currently preferred as it results in the substantial purification of the mutacin, as assessed by deferred antagonism, in yields sufficient for further characterization and use. This preferred mode of mutacin polypeptide purification involves the execution of certain purification steps in the order described herein. However, as is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified antimicrobial mutacin polypeptide.
As mentioned above, although preferred for use in certain embodiments, there is no general requirement that the mutacin always be provided in their most purified state. Indeed, it is contemplated that less substantially purified mutacin, which are nonetheless substantially enriched in antimicrobial activity relative to the natural state, will have utility in certain embodiments. These embodiments include using the mutacin as a diagnostic test for S. mutans subtypes. Serologic reagents developed against the mutacin may also be used to rapidly identify and define mutacin subtypes. Partially purified mutacin polypeptide fractions for use in such embodiments may be obtained by subjecting a bacterial extract to one or a combination of the steps described above.
Using the present purification procedures the inventors have produced and purified mutacin from two strains of S. mutans: T8 and UA96. Although similar in their basic characteristics such as molecular weight, resistance to heat and pH, and solubility in water immiscible organic solvents, minor differences in the mutacin have been noted between these two closely related strains. For example, the mutacins exhibit different migration patterns on TLC. Using a degenerate oligonucleotide complementary to the amino terminal mutacin protein sequence polymorphisms are apparent in Southern hybridization patterns. These minor differences are also clear using arbitrary primer-PCR fingerprinting. Finally, the mutacin derived from the S. mutans T8 strain was more amenable to purification which allowed the determination of molecular weight by mass spectroscopy and protein sequencing. Naturally it will understood by those of skill in the art that mutacins with such variations as these, which nevertheless have the general properties described forthwith fall within the scope of the present invention.
Further compositions of the invention include pharmaceutical composition comprising substantially purified mutacin polypeptides dispersed in pharmacologically acceptable carriers. The admixture consisting essentially of a therapeutically effective amount of the mutacin polypeptide in a pharmacologically acceptable carrier. These carriers may be incorporated with excipients and used in the form of, e.g., mouthwashes, dentifrices, assimilable edible carriers, tablets, buccal tables, trouches, capsules, elixirs, wafers, suspensions, syrups, and the like as disclosed herein, and by means well known by those of skill in the art. The mutacin may also be incorporated into pharmacologically acceptable carriers for parenteral delivery, such as suspensions used for intravenous, intramuscular and subcutaneous injection. The phrase xe2x80x9cpharmaceutically acceptablexe2x80x9d refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
The preparation of an aqueous composition that contains a mutacin as an active ingredient will be well understood to those of skill in the art in light of the present disclosure. Typically, such compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
A technique often employed by those skilled in the art of protein production today is to obtain a so-called xe2x80x9crecombinantxe2x80x9d version of the protein, to express it in a recombinant cell and to obtain the protein from such cells. These techniques are based upon the xe2x80x9ccloningxe2x80x9d of a DNA molecule encoding the protein from a DNA library, i.e., on obtaining a specific DNA molecule distinct from other portions of DNA. This can be achieved by, for example, cloning a cDNA molecule, or cloning a genomic-like DNA molecule. Techniques such as these would also, of course, be appropriate for the production of the mutacin polypeptides in accordance with the present invention.
A first step in such cloning procedures is the screening of an appropriate DNA library, such as, in the present case, an S. mutans-derived library. The screening procedure may be an expression screening protocol employing antibodies directed against the protein, or activity assays. Alternatively, screening may be based on the hybridization of oligonucleotide probes or PCR amplification using the primers designed from a consideration of portions of the amino acid sequence of the protein, or from the DNA sequences of genes encoding related proteins. Another cloning approach particularly suitable is the use of a probe or primer directed to a gene known to be generally associated with, e.g., within the same operon as, the structural gene that one desires to clone. For example, in the case of mutacin, one may wish to use a primer directed to the conserved ABC transporter genes that are associated with all lanthionine antibiotic genes.
After identifying an appropriate DNA molecule by any or a combination of means as described above, the DNA may be then inserted into any one of the many vectors currently known in the art and transferred to a prokaryotic or eukaryotic host cell where it will direct the expression and production of the so-called xe2x80x9crecombinantxe2x80x9d version of the protein. The recombinant host cell may be selected from a group consisting of S. mutans, E. coli, S. cerevisae, Bacillus sp., Lactococci sp., Enterococci sp., or Salmonella sp. In certain preferred embodiments, the recombinant host cell will have a RecAxe2x88x92 phenotype.
The recombinant mutacin may differ from naturally-produced mutacin in certain ways. In particular, the degree of post-translational modifications, such as, for example, glycosylation and phosphorylation or dehydration and transformation of thioether bridges may be different between the recombinant mutacin and the mutacin polypeptide purified from a natural source, such as S. mutans T8 or UA96.
In still another embodiment, the present disclosure provides methods for cloning the DNA encoding the mutacin polypeptide. Using methods well known to those of skill in the art, the DNA that encodes the purified S. mutans mutacin of the present invention may be isolated and purified. For example, by designing a degenerate oligonucleotide comprising nucleotides complementary to the peptide sequences (SEQ ID NO:1 and 2) the mutacin encoding DNA can be cloned from an S. mutans genomic DNA library.
The DNA sequences disclosed by the invention allow for the preparation of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to S. mutans gene sequences encoding the present antibiotic, termed mutA. The term mutA as used herein is meant to describe the gene locus encoding the mutacin structural gene. In these aspects, nucleic acid probes of an appropriate length are prepared. Such probes are typically prepared based on the consideration of the defined amino acid sequences of purified mutacin. The ability of such nucleic acid probes to specifically hybridize to the S. mutans mutA gene sequences lend them particular utility in a variety of embodiments. For example, the probes may be used in a variety of diagnostic assays for detecting the presence of S. mutans organisms in a sample, for example, a saliva sample from the oral cavity. However, other uses are envisioned, including identification and isolation of mutA gene sequences encoding similar or mutant polypeptides related to the mutacin. Other uses include the use of mutant species primers or primers to prepare other genetic constructs.
In further embodiments, the DNA encoding the mutacin of the present invention allows for the large scale production and isolation of the mutacin polypeptide. This can be accomplished by directing the expression of the mutacin polypeptide by cloning the DNA encoding the mutacin polypeptide into a suitable expression vector. Such an expression vector may then be transformed into a host cell that is able to produce the mutacin protein. The mutacin protein may then be purified, e.g., by means provided for in this disclosure and utilized in a biologically active form. Non-biologically active recombinant mutacin may also have utility, e.g., as an immunogen to prepare anti-mutacin antibodies.
Another approach toward identifying the gene(s) responsible for the production of mutacin is to locate genes known to be adjacent to the mutacin structural gene. From sequenced lantibiotic gene loci, it is clear that several processing and export enzymes are highly conserved among the lantibiotic producers and share areas of common sequences. For example, all the known lantibiotic producers have so-called ABC transporter genes responsible for making a protein involved with mutacin export. A series of oligonucleotide primers complementary to conserved sequences could be used in PCR reactions to amplify the intervening sequence, this amplicon could be used as a probe to identify the ABC transporter gene (PCR technology is described in U.S. Pat. No. 4,603,102). Since the ABC transporter gene is part of every known lantibiotic loci described thus far, the structural gene for mutacin should be nearby and readily identified by so called xe2x80x9cchromosome walkingxe2x80x9d.
Methodological aspects of the invention include methods for contacting gram-positive bacteria with the mutacin polypeptides, thereby killing the bacteria. The term xe2x80x9ccontacting gram-positive bacteriaxe2x80x9d as used herein, is meant to encompass the delivery of an amount of mutacin having a substantial bacteriocidal component. The xe2x80x9ccontactxe2x80x9d process is the process by which the mutacin polypeptide comes in direct juxtaposition with the target cell. The term xe2x80x9ctarget cellxe2x80x9d as used herein, is a bacterial cell against which the present mutacin exhibits substantial bacteriocidal activity.
To contact mutacin polypeptides with the target cells with a mutacin-containing composition one may simply add the polypeptides or composition to potential bacterial targets in vitro. Alternatively, one may administer a biologically effective amount of a pharmacologically acceptable form of the mutacin polypeptides or composition to an animal, where it will contact target cells in a biological fluid in vivo. In this context, xe2x80x9ccontactxe2x80x9d is achieved simply by administering the composition to the animal.
Virtually any pharmaceutical polypeptide formulation may be used, including, but not limited to, formulations for parenteral administration, such as for intravenous, intramuscular and subcutaneous administration; inhalants, aerosols and spray formulations; formulations of peptides for topical use, such as in creams, ointments, dentifrices, powders, and gels; polypeptides encapsulated in micelles or liposomes and drug release capsules including the active peptides incorporated within a biocompatible coating designed for slow-release; and mouthwashes and other washes.
In another preferred embodiment, a pharmacological composition containing the mutacin protein of the present disclosure may be used for the treatment of dental caries. The treatment comprising orally administering a therapeutically effective amount of the mutacin polypeptide in a pharmacologically acceptable carrier. In another embodiment the polypeptide is administered topically. The mutacin polypeptides may be used for preventing or inhibiting development of dental caries by administering to an individual having or susceptible to dental caries a therapeutically effective amount of a composition comprising the mutacin polypeptide in a pharmacologically acceptable carrier. The genes responsible for mutacin production may also be incorporated into another member of the oral cavity flara and expressed.